Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. ...These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Translating whole exome sequencing (WES) for prospective clinical use may impact the care of cancer patients; however, multiple innovations are necessary for clinical implementation. These include: ...(1) rapid and robust WES from formalin-fixed paraffin embedded (FFPE) tumor tissue, (2) analytical output similar to data from frozen samples, and (3) clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival FFPE tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a “long tail” of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15/16 patients. In one patient, previously undetected findings guided clinical trial enrollment leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become ...increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.
The conservation status of small cetaceans has significantly worsened since the 1980s, when the baiji was the only species of small cetacean listed as Endangered by IUCN. Now the baiji is almost ...certainly extinct and 13 other species, subspecies, or populations (hereafter units-to-conserve or units) of small cetaceans are listed as Critically Endangered (CR) on the IUCN Red List. Bycatch is the main threat to 11 of the CR units. Entanglement in gillnets contributed to the extinction of the baiji and is responsible for the imminent extinction of the vaquita. Unfortunately, there is no simple technical solution to the problem of bycatch of small cetaceans. If the 8 CR units with 100 or fewer remaining individuals are to be saved, conservation zones must be established where gillnets are eliminated and bans on their use are strictly enforced. Recent experience with the vaquita in Mexico demonstrates that enforcement of such conservation zones can be very difficult. Ineffective enforcement is also a problem for at least 4 of the other CR units. Time is very short and, unless major efforts are made now to address the bycatch problem, the prospects for CR small cetaceans and other at-risk aquatic megafauna are grim. The ultimate long-term solution to the bycatch problem is the development of efficient, inexpensive, alternative fishing gear that can replace gillnets without jeopardizing the livelihoods of fishermen. Good fishery governance and the direct involvement of fishing communities are also essential to the successful conservation of most threatened populations of small cetaceans.
Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association ...studies.
We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV. Using long-insert jumping libraries at 105X mean physical coverage and linked-read whole-genome sequencing from 10X Genomics, we document seven major SV classes at ~5 kb SV resolution. Our results encompass 11,735 distinct large SV sites, 38.1% of which are novel and 16.8% of which are balanced or complex. We characterize 16 recurrent subclasses of complex SV (cxSV), revealing that: (1) cxSV are larger and rarer than canonical SV; (2) each genome harbors 14 large cxSV on average; (3) 84.4% of large cxSVs involve inversion; and (4) most large cxSV (93.8%) have not been delineated in previous studies. Rare SVs are more likely to disrupt coding and regulatory non-coding loci, particularly when truncating constrained and disease-associated genes. We also identify multiple cases of catastrophic chromosomal rearrangements known as chromoanagenesis, including somatic chromoanasynthesis, and extreme balanced germline chromothripsis events involving up to 65 breakpoints and 60.6 Mb across four chromosomes, further defining rare categories of extreme cxSV.
These data provide a foundational map of large SV in the morbid human genome and demonstrate a previously underappreciated abundance and diversity of cxSV that should be considered in genomic studies of human disease.
Bacterial keratitis is a serious and vision-threatening condition in veterinary and human patients, one that often requires culture and susceptibility testing to adjust therapy and improve clinical ...outcomes. The present study challenges the antimicrobial susceptibility testing (AST) paradigm in ophthalmology, enabling more accurate
-to-
translation by incorporating factors normally present during host-pathogen interactions in clinical patients. Thirty bacteria (10
, 10
, 10
) were isolated from canine patients with infectious keratitis. For each isolate, commercial plates (Sensititre™ JOEYE2) were used to assess the minimal inhibitory concentration (MIC) of 17 different antibiotics in the absence (0% albumin, control) or presence of canine albumin (0.01-2%). For
, the experiment was repeated with actual tear fluid collected from canine eyes with ocular surface inflammation. Kruskal-Wallis, Wilcoxon signed rank test and Spearman's correlation tests were used for statistical analysis. Clinical outcomes were unfavorable in selected canine patients with bacterial keratitis (e.g., globe perforation, graft dehiscence) despite standard AST (i.e., 0% albumin in test medium) confirming that most corneal infections (93%) were susceptible to ≥1 topical antibiotics used at the initial visit. Albumin levels ≥0.05% increased MICs in a dose-dependent, bacteria-specific, and antibiotic-specific manner. No significant differences (
= 1.000) were noted in MICs of any antibiotic whether albumin or tear fluid was added to the Mueller-Hinton broth. Percent protein binding inherent to each antibiotic was associated with clinical interpretations (Spearman's rho = -0.53,
= 0.034) but not changes in MICs. Albumin in tears impacted the efficacy of selected ophthalmic antibiotics as only the unbound portion of an antibiotic is microbiologically active. The present findings could improve decision making of clinicians managing bacterial keratitis, reduce development of antimicrobial resistance, influence current guidelines set by the Clinical and Laboratory Standards Institute, and serve as a reference for bacteriological evaluations across medical fields and across species.
Abstract Objective To describe features of maternal concern for her child undereating; examine maternal and child correlates of maternal concern for undereating; and determine whether maternal ...concern for undereating is associated with feeding practices. Methods This was a cross-sectional analysis of an observational study with 286 mother–child dyads (mean child age, 71 months). Maternal concern for undereating was assessed using a semistructured interview. Mothers completed questionnaires to assess picky eating, food neophobia, and feeding practices. Feeding practices were further assessed using videotaped mealtime observations. Logistic regression was used to assess the association of maternal and child characteristics with maternal concern for undereating. Regression was used to assess the association of maternal concern for undereating with feeding practices, controlling for covariates. Results Over a third of mothers (36.5%) expressed concern that their child does not eat enough. Correlates of concern for undereating included child body mass index z-score (BMIz; odds ratio OR = 0.58; 95% confidence interval CI, 0.43–0.77) and picky eating (OR = 2.41; 95% CI, 1.26–4.59). Maternal concern for undereating was associated with greater reported pressure to eat (relative risk RR = 1.97; 95% CI, 1.55–2.50), greater observed bribery (OR = 2.63; 95% CI, 1.50–4.60), and higher observed pressure (OR = 1.90; 95% CI, 1.08–3.36) during mealtimes. Conclusions Mothers of children who are picky eaters and have a lower BMIz are more likely to be concerned that their children do not eat enough, and maternal concern for undereating is associated with pressuring and bribing children to eat. Pediatricians might address maternal concern for undereating by advising feeding practices that do not involve pressure and bribery, particularly among healthy weight children.
Picky eating is common in children. Few studies have examined predictors of picky eating, and the association of picky eating with weight status and dietary quality is inconsistent in the literature. ...We aimed to identify predictors of picky eating and to test the association of picky eating with child body mass index z-score (BMIz), dietary quality, and micronutrient intake.
This was a cross-sectional analysis of baseline data from a randomized controlled trial to prevent obesity among 506 preschoolers attending Head Start. Parents completed questionnaires to assess picky eating and child temperament. Three 24-hour dietary recalls were collected to assess dietary intake. Multivariate regression models assessed child, parent, and family predictors of picky eating; additional models tested adjusted associations of picky eating with child BMIz, dietary quality (measured using the Healthy Eating Index-2010), and micronutrient intake.
Picky eating was predicted by male sex, older child age, and more difficult temperament but not race/ethnicity, maternal body mass index, maternal depressive symptoms, household food insecurity, or single parent home. Picky eating was not associated with child BMIz or micronutrient deficiencies; it was inversely associated with total Healthy Eating Index-2010 score and servings of whole fruit, total vegetables, greens and beans, and total protein foods.
Pediatric providers should support parents in expanding the number of healthy foods the child eats to improve dietary quality, but reassure parents that picky eating is not associated with children's weight status or micronutrient deficiencies.
Treatment of BRAF-mutant melanoma with combined dabrafenib and trametinib, which target RAF and the downstream MAP-ERK kinase (MEK)1 and MEK2 kinases, respectively, improves progression-free survival ...and response rates compared with dabrafenib monotherapy. Mechanisms of clinical resistance to combined RAF/MEK inhibition are unknown. We performed whole-exome sequencing (WES) and whole-transcriptome sequencing (RNA-seq) on pretreatment and drug-resistant tumors from five patients with acquired resistance to dabrafenib/trametinib. In three of these patients, we identified additional mitogen-activated protein kinase (MAPK) pathway alterations in the resistant tumor that were not detected in the pretreatment tumor, including a novel activating mutation in MEK2 (MEK2(Q60P)). MEK2(Q60P) conferred resistance to combined RAF/MEK inhibition in vitro, but remained sensitive to inhibition of the downstream kinase extracellular signal-regulated kinase (ERK). The continued MAPK signaling-based resistance identified in these patients suggests that alternative dosing of current agents, more potent RAF/MEK inhibitors, and/or inhibition of the downstream kinase ERK may be needed for durable control of BRAF-mutant melanoma.
We present genetic and morphological evidence supporting the recognition of a previously synonymized species of Mesoplodon beaked whale in the tropical Indo‐Pacific, Mesoplodon hotaula. Although the ...new species is closely‐related to the rare ginkgo‐toothed beaked whale M. ginkgodens, we show that these two lineages can be differentiated by maternally (mitochondrial DNA), biparentally (autosomal), and paternally (Y chromosome) inherited DNA sequences, as well as by morphological features. The reciprocal monophyly of the mtDNA genealogies and the largely parapatric distribution of these lineages is consistent with reproductive isolation. The new lineage is currently known from at least seven specimens: Sri Lanka (1), Gilbert Islands, Republic of Kiribati (1+), Palmyra Atoll, Northern Line Islands, U.S.A. (3), Maldives (1), and Seychelles (1). The type specimen (Sri Lanka) was described as a new species, M. hotaula, in 1963, but later synonymized with M. ginkgodens. This discovery brings the total number of Mesoplodon species to 15, making it, by far, the most speciose yet least known genus of cetaceans.
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